1. Technical Field
The field of this invention is related to the preparation of human monoclonal. antibodies (HMab) to structurally conserved epitopes of HCV found in a high proportion of patients for diagnosis and therapy and the ability to distinguish genotype by the use of antibodies.
2. Background
Hepatitis C virus (HCV) is an enveloped virus the genetic information for which is encoded in a 9.5 kb positive strand RNA genome. A highly conserved noncoding region of 341 bp is localized at the 5xe2x80x2-end of this viral genome, which is followed by a long open-reading frame coding for a polyprotein of approximately 3,010 amino acids. Two putative envelope glycoproteins E1 (gp35) and E2 (gp72) have been identified with 5 or 6 and 11 N-linked glycosylation sites, respectively. A high level of genetic variability is associated with the envelope genes. This is highly accentuated at the 5xe2x80x2- end of the E2 gene, where two hypervariable regions termed HVR1 and HVR2, have been described. Antibodies to HVR1 appear to mediate virus neutralization in cell culture and chimpanzee protection studies (Farci, et al., 1996 Proc Natl Acad Sci USA. 93:15394-15399; Shimizu, et al., 1994 J Virol. 68:1494-1500). Unfortunately, antibodies to HVR1 tend to be isolate specific and over time drive the replication of new viral variants that the existing immune response does not recognize (Farci, et al, 1994 Proc Natl Acad Sci USA. 91:7792-7796; Weiner, et al., 1992 Proc Natl Acad Sci USA. 89:3468-3472; Kato, et al., 1993 J Virol. 67:3923-3930), although progress has been made at inducing a broader immune response to HVR1 related sequences (Puntoriero, et al., 1998 EMBO Journal 17:3521-3533). HCV envelope antigens appear to be highly immunogenic when expressed in glycosylated forms (da Silva Cardoso, et al., 1997 Ann. Hematol. 74:135-7). Preliminary data suggest the existence of conserved epitopes within the E2 protein (Lesniewski, et al., 1995 J. Med. Virol. 45:415-22). The existence of neutralizing antibodies in serum from infected patients has been proposed.
Studies using HCV E1-E2 proteins expressed in mammalian cells showed that infected individuals have an antibody response to HCV E2 composed in part to epitopes that are conformational in nature (Harada, et al., 1994 J Gen. Virol. 76:1223-1231). Studies involving the isolation of human monoclonal or recombinant antibodies to HCV E2 protein showed that a substantial fraction of these antibodies recognize conformational epitopes (da Silva Cardoso et al., 1998 J. Med Virol. 55:28-34; Burioni et al., 1998 Hepatology 28:810-814; Habersetzer et al., (1998) Virology 249:32-41). As to biological function of these domains, investigators have employed surrogate assays to provide insights into virus neutralization since the virus cannot be grown, in vitro (Houghton. Hepatitis C viruses. In Fields B N, Knipe D M, Howley P M (eds) Virology. Lippincott-Raven, Philadelphia, pp 1035-1058). One surrogate assay, the neutralization of binding (NOB) assay, evaluates the ability of a given antibody or serum to prevent the association of HCV E2 protein with a human T-cell line (Rosa, et al., 1996 Proc Natl Acad Sci USA. 93:1759-1763). The finding that serum antibodies obtained from chimpanzees protected by vaccination were strongly positive in the NOB assay provides support for the relevance of the assay as a measure of virus neutralization activity (Rosa, et al., supra; Ishii, et al., 1998 Hepatology 28:1117-1120).
The human tetraspannin cell surface protein CD81 (TAPA-1, for review see Levy, et al., 1998 Ann. Rev. Immunol. 16:89-109) is the target protein bound by HCV E2 in the NOB assay (Pileri, et al., 1998 Science. 282:938-941). Furthermore, human CD81 binds to free virions, and subsequently is a possible receptor for HCV (Pileri, et al., supra). Using HCV 1a E2 proteins, several human monoclonal antibodies to HCV E2 protein have been reported to inhibit the interaction of HCV E2 with human cells (Burioni, et al., 1998 Hepatology 28:810-814; Habersetzer, et al., 1998 Virology 249:32-41). However, little is known about the conservation of the epitopes recognized by the NOB positive antibodies in HCV E2 proteins of different genotypes.
Other approaches to detection of and protection against HCV include the development of peptide mimetics. As an example, peptide mimetics of Hepatitis type A and C viral proteins have been created through production of randomly generated synthetic and phage-display peptide libraries for use in detection assays and vaccination therapies (Mattioli, et al., 1995 J Virology 69:5294-5299 and Prezzi, et al., 1996 J Immunol. 156:4504-4513). However, effective antibody binding of these mimotopes has only been compared to linearly defined viral epitopes. The sequential recombinant fusing of several linearly defined immunodominant HCV epitopes has been described for use in diagnostic assays (Chein, et al., 1999 J Clin Microbiol. 37:1393-1397). However, this multiple-epitope fusion antigen designed from linear epitopes was not created to function in the same capacity as a conformational mimetic: it was not designed to interfere with binding to a target receptor.
It is therefore of substantial interest to identify neutralizing antibodies in serum from infected patients which may be used in diagnosis and passive immunotherapy, where the antibodies would originate from a human cell, and provide for neutralization of a broad spectrum of genotypes, particularly in a particular geographical area. Both breadth of reactivity to multiple HCV genotypes and the ability to interfere with the binding of HCV virions to susceptible cells would be key attributes for a therapeutically useful neutralizing antibody. Also of interest is the design of peptide and non-peptide (inorganic) structural mimetics of HCV envelope proteins.
Relevant Literature
References providing background information concerning HCV include Abrignani 1997 Springer Semin. Immunopathology 19:47-55; Simmonds, 1995 Hepatology 21:570-583; and Mahaney et al., 1994 Hepatology 20:1405-1411.
da Silva Cardosa et al., 1998 J. of Med. Virology 55:28-34 describe human monoclonal antibodies to HCV E1/E2. Habersetzer et al., 1998 Virology 249:32-41 describe human monoclonal antibodies capable of recognizing HCV E2 genotypes 1a and 1b. Burioni et al., 1998 report human recombinant Fabs for the HCV E2 protein. Deleersnyder et al., 1997 J. of Virology 71:697-704 describe an E2 reactive monoclonal antibody. Other references related to the use of antibodies to HCV include Burioni et al., 1998 Hepatology 28:810-814; Akatsuka, et al., 1993 Hepatology 18:503-510; DeLalla, et al., 1993 J. Hepatol. 18:163-167; Mondelli, et al., 1994 J. Virol. 68:4829-4836; Siemoneit, et al., 1994 Hybridoma 13:9-13; and Moradpour, et al., 1996 J. Med. Virol. 48:234-241; for producing human antibodies, Foung, et al., 1990 J. Immunol. Methods 70:83-90; Zimmerman, et al., 1990 J. Immunol. Methods 134:43-50;for producing modified antibodies using combinatorial libraries, Burton and Barbas, Dixon, F J (Ed.) Advances in Immunology, Vol. 57, Vi+391 p. Academic Press, Inc., San Diego, Calif. 191-280, 1994; Plaisant, et al., 1997 Res. Virol. 148-169; and Barbas and Burton, Monoclonal Antibodies from Combinatorial Libraries. Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor, N.Y., 1994.
An assay for antibodies binding to HCV E2 is described by Rosa, et al., 1996 Proc Natl Acad Sci USA 93:1759-1763.
Vaccinia virus or baculovirus constructs having a portion of the HCV genome are described by Ralston et al., 1993 J. of Virology 67:6733-6761 and Lanford et al., 1993 Virology 197:225-235.
Monoclonal antibodies, including human monoclonal antibodies, are provided which bind to the dominant HCV types in major geographical areas. Specifically, a family of monoclonal antibodies binding to conformationally conserved epitopes of the HCV E2 protein are provided. Among the family are antibodies which bind to the dominant genotypes found in the United States, so as to be substantially pan-monoclonal antibodies in being able to bind to almost all cases of HCV infection which have been diagnosed in the United States, as well as at least a substantial proportion of the cases in other geographic locales. The monoclonal antibodies find use in a variety of diagnostic assays. In addition, conformationally conserved expression of recombinant type 1 and type 2 HCV E2 proteins are provided for use in assays, screening drugs and for other purposes. The human antibodies find use in passive immunotherapy strategies for reducing viral load of infected individuals and interfering with infection of target cells. Antibodies recognizing conformationally dependent epitopes can also be used to provide a template for the rational design of peptide and inorganic conformationally-defined epitope mimetics.